X-ray Crystallography: A Very Brief History
Macromolecular X-ray crystallography is a structural biology technique used to determine the atomic structure of macromolecules such as proteins, DNA, and RNA. The origins of X-ray crystallography begin in 1912, when Max von Laue exposed a copper sulfate crystal to X-rays to produce a diffraction pattern. This proved the theory X-rays are electromagnetic waves, demonstrated that the regular spacing of the atoms in a crystal acts as a diffraction grating, and provided direct evidence for the — earning von Laue the .
Building on this work the father-son duo William and Lawrence Bragg were responsible for the birth of X-ray crystallography in 1913. Using an X-ray spectrometer built by William Bragg and Bragg's law of X-ray diffraction determined by Lawrence Bragg, the pair solved the crystal structures of sodium chloride, potassium chloride, potassium bromide and potassium iodide in their paper, "". In 1915 they were both awarded the Nobel Prize in Physics, making Lawrence the youngest person ever to receive the accolade.
It would be many years until the first protein structure was solved, a milestone achieved by John Kendrew in 1958 with the determination of , a feat 25 years in the making that heralded the beginning of Protein Crystallography. In the modern era, macromolecular crystallography has become an indispensable tool for biologists to determine the structure of significantly more complex proteins and protein complexes. The considerable evolution in computing power, synchrotrons, detectors, molecular biology, and robotics mean structure solution is easier today than it has ever been, but still not a given. With a sufficiently pure and homogeneous protein sample, any researcher can enter the crystallographic pipeline and determine high-resolution structures.
The prolific work of structural biologists for the past 60+ years has resulted in over 200,000 structures being deposited into the (PDB). This vast repository has fuelled the development of predictive AI tools such as AlphaFold, now in its .
Crystallisation
Considerable advances in the molecular biology of and the addition of affinity tags to proteins of interest mean that crystallisation has now become the major bottleneck in the protein crystallography pipeline. Protein crystallisation is achieved by addition of mild precipitation agents to purified protein solution in the right concentration to produce a supersaturated state which encourages the macromolecules to fall out of solution and form a crystal.
The most common method for macromolecular crystallisation is vapour diffusion. In this method the protein and precipitant are mixed in a sealed environment above an excess of the precipitation solution, also known as mother liquor or well solution. Over time the protein/precipitate drop and well solution equilibrate, with water diffusing from the mixed drop to the well solution. This causes an increase in the protein and precipitant concentration, which can lead to nucleation of protein crystals. As crystals form, the soluble protein concentration decreases. This halts further nucleation and allows existing crystals to grow. These crystallisation trajectories are best explained through use of a phase diagram, such as those produced in and .
Finding the right condition to form crystals can be challenging and sample intensive. However, the development of robotics and commercial screens which cover a broad range of chemical space has simplified the process of finding and optimising these conditions for your protein while keeping sample consumption to a minimum.
For a more thorough introduction to protein crystallisation, we recommend the tutorials put together by from Uppsala University in Sweden and this Introduction to Protein Crystallisation from .
Preparing Crystals for Diffraction Experiments
Modern X-ray sources produce high-intensity X-ray beams which cause significant radiation damage to biological molecules during data collection. To mitigate this damage the majority of protein crystal diffraction data are collected at cryogenic temperatures (100 K) by flash cooling the crystals in liquid nitrogen. This increases the lifetime of the crystals in the X-ray beam to allow complete datasets, or even multiple datasets, to be collected from a single protein crystal. The major downside is the need to cryoprotect the protein crystals to prevent formation of ice crystals which can degrade diffraction. A number of common laboratory chemicals can act as cryoprotectants (glycerol, PEG 400, ethylene glycol, MPD, sucrose), but experimentation may be required to find the right one for your crystallisation condition.
Room Temperature Data Collection
Recent advances in detector speed and the development of new data collection techniques, such as the in-situ beamline and , now make room-temperature data collection routine. This is particularly useful if you are interested in studying the dynamic properties of your protein which would be rendered immobile under cryogenic data collection.
Data processing
Processing X-ray diffraction data has never been more accessible, with the development of that perform data reduction and phase determination. It is now possible to generate an initial structural model with minimal prior crystallography experience. Should the automatic pipelines fail, the facility manager is available to provide guidance and training on manual data reduction, structure determination and refinement.